Hyperblebbing shigella strains

ABSTRACT

Hyperblebbing  Shigella  strains are generated by disrupting one or more components of the Tol-Pal system. The blebs from these strains are useful immunogens for vaccination. The individual proteins found in these blebs can also be used as immunogens.

TECHNICAL FIELD

This invention is in the field of immunisation against Shigella species.

BACKGROUND ART

Shigella are Gram-negative non-motile facultative anaerobic bacilli that fall into four serogroups: S. dysenteriae, S. flexneri, S. boydii and S. sonnies. They cause shigellosis (bacillary dysentery).

The hallmark of clinical shigellosis is an acute rectocolitis associated with fever, nausea, anorexia, dehydration, mucopurulent and bloody diarrhea, and tenesmus. Shigella-caused dysentery is endemic and causes millions of illness episodes in developing countries. For example, there are estimated to be 165 million cases of Shigella diarrhea per year, 99% of which occur in developing countries and 69% of which occur in children under five years of age. The morbidity and mortality due to shigellosis are especially high among children in developing countries.

Existing approaches to Shigella vaccines were reviewed in ref 1 and have been based on live attenuated strains for oral immunisation, conjugated 0 saccharides for injection, proteosomes (meningococcal outer membrane vesicles with attached Shigella LPS) for intranasal use, invaplexes (subcellular extracts of Shigella including IpaB, IpaC and LPS) for intranasal use, and nuclear protein-ribosomal complexes prepared from msbB^(−ve) strains with detoxified LPS. Although some of these vaccines have been efficacious in field trials, none protects against multiple Shigella serotypes.

It is an object of the invention to provide further and improved components useful in preparing Shigella vaccines, and in particular vaccines which can protect against multiple serotypes.

DISCLOSURE OF THE INVENTION

Shigella spontaneously release outer membrane blebs during growth due to the turgour pressure of the cell envelope. As disclosed in reference 2, release of the blebs is highly dependent on the bacterial envelope structure. The inventors have used a mutant strain of Shigella in which the Tol-Pal system has been disrupted to disturb the envelope structure. During normal growth these mutant strains release into their culture medium large quantities of blebs which are rich in immunogenic outer membrane proteins, and these blebs can thus be used as immunogens.

Thus the invention provides a Shigella bacterium which expresses no more than 4 of TolA, TolB, TolQ, TolR and Pal proteins. Thus at least one protein from the natural five-protein Tol-Pal system is absent, resulting in a bacterium which, during growth in culture medium, releases greater quantities of outer membrane blebs into the medium than the same bacterium expressing all 5 Tol-Pal proteins. Preferably TolR is not expressed, but the other four proteins may be expressed.

The invention also provides a Shigella bacterium which does not express a TolR protein. The invention also provides a ΔtolR strain of Shigella, such as a ΔtolRΔgalU strain.

The invention also provides a Shigella bacterium which expresses TolA, TolB, TolQ, TolR and Pal proteins, wherein the TolA, TolQ, TolR and/or Pal protein (a) is located in the bacterium's inner or outer membrane, and (b) includes one or more amino acid sequence mutation(s) such that, compared to the same bacterium without said mutation(s), the bacterium releases greater quantities of outer membrane blebs when growing in culture medium.

The invention also provides a Shigella bacterium in which one or more components of its Tol-Pal system has a modification such that, during growth in culture medium, the bacterium releases greater quantities of outer membrane blebs into the medium than the same bacterium lacking the modification, and which does not express: (i) a native Shigella lipopolysaccharide and/or (ii) a Shigella enteric toxin.

The invention also provides a method of preparing a hyperblebbing Shigella bacterium, comprising a step of modifying gene(s) encoding one or more components of a starting bacterium's Tol-Pal system such that the modification causes the bacterium, when grown in culture medium, to release greater quantities of outer membrane blebs into the medium than the starting bacterium, and wherein the modification involves mutating one or more of the starting bacterium's tolA, to1B, tolQ, tolR and/or pal genes. The mutating step may delete the gene. The method may also involve modification of gene(s) encoding a protein required for synthesis of the bacterium's lipopolysaccharide or an enteric toxin.

The invention also provides a bleb isolated or obtainable from a bacterium of the invention. These blebs are useful as components of Shigella vaccines.

The invention also provides a process for preparing Shigella blebs, comprising a step of separating the blebs from a culture medium comprising bacteria of the invention which have been grown under conditions which permit the release of blebs into the medium by the bacteria. Blebs prepared by this process can be used as components of Shigella vaccines.

The invention also provides a culture medium comprising bacteria of the invention which have been grown under conditions which permit the release of blebs into the medium by the bacteria. Blebs may be purified from this culture medium.

The invention also provides a composition comprising blebs that, during culture of bacteria of the invention, are released into the culture medium. This composition does not comprise any living and/or whole bacteria. This composition and/or its components can be used for Shigella vaccine preparation.

The invention also provides a composition comprising blebs, wherein the blebs are present in the filtrate obtainable after filtration through a 0.22 μm filter of a culture medium in which a bacterium of the invention has been grown. This composition and/or its components can be used for Shigella vaccine preparation. The invention also provides a Shigella bleb which includes one or more (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60) of: (a) a protein consisting of an amino acid sequence selected from SEQ ID NOs: 8 to 67; (b) a protein comprising an amino acid sequence having at least j % identity to one of SEQ ID NOs: 8 to 67, where j is 50 or more (e.g. 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99) and/or comprising a fragment of at least n consecutive amino acids of any one of SEQ ID NOs: 8 to 67, wherein n is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments comprise an epitope from one of SEQ ID NOs: 8 to 67. Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of the SEQ ID NO: while retaining at least one epitope of the SEQ ID NO:. Other fragments omit one or more protein domains e.g. lacking a signal peptide, etc. 60 proteins have been confirmed as present within blebs of the invention and to be immunoreactive with sera prepared against the blebs. Thus the individual proteins may be used as immunogenic components in purified form, separate from the blebs. Thus the invention also provides a bleb-free immunogenic composition comprising a bleb protein comprising: (a) one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60) of amino acid sequences SEQ ID NOs 8 to 67; (b) an amino acid sequence having at least j % identity to one of SEQ ID NOs: 8 to 67, where j is 50 or more (e.g. 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99) and/or comprising a fragment of at least n consecutive amino acids of any one of SEQ ID NOs: 8 to 67, wherein n is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments comprise an epitope from one of SEQ ID NOs: 8 to 67, and more preferred fragments are immunogenic fragments. Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of a SEQ ID NO: while retaining at least one epitope of the SEQ ID NO:. Other fragments omit one or more protein domains e.g. lacking a transmembrane domain, a signal peptide, etc.

The invention also provides a Shigella bleb which includes one or more (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128 or 129) of: (a) a protein consisting of an amino acid sequence selected from SEQ ID NOs: 8 to 136; (b) a protein comprising an amino acid sequence having at least j % identity to one of SEQ ID NOs: 8 to 136, where j is 50 or more (e.g. 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99) and/or comprising a fragment of at least n consecutive amino acids of any one of SEQ ID NOs: 8 to 136, wherein n is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments comprise an epitope from one of SEQ ID NOs: 8 to 136. Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of the SEQ ID NO: while retaining at least one epitope of the SEQ ID NO:. Other fragments omit one or more protein domains e.g. lacking a signal peptide, etc.

129 proteins have been confirmed as present within blebs of the invention and to be immunoreactive with sera prepared against the blebs. Thus the individual proteins may be used as immunogenic components in purified form, separate from the blebs. Thus the invention also provides a bleb-free immunogenic composition comprising a bleb protein comprising: (a) one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128 or 129) of amino acid sequences SEQ ID NOs 8 to 136; (b) an amino acid sequence having at least j % identity to one of SEQ ID NOs: 8 to 136, where j is 50 or more (e.g. 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99) and/or comprising a fragment of at least n consecutive amino acids of any one of SEQ ID NOs: 8 to 136, wherein n is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments comprise an epitope from one of SEQ ID NOs: 8 to 136, and more preferred fragments are immunogenic fragments. Other preferred fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of a SEQ ID NO: while retaining at least one epitope of the SEQ ID NO:. Other fragments omit one or more protein domains e.g. lacking a transmembrane domain, a signal peptide, etc.

Within SEQ ID NOs: 8 to 136, a preferred subset in relation to S. flexneri is the SEQ ID NOs: listed in “Subset 1” beneath Table 1. Within SEQ ID NOs: 8 to 136, a preferred subset in relation to S. sonnies is the SEQ ID NOs: listed in “Subset 2” beneath Table 1

The Tol-Pal system

Like many Gram-negative bacteria, the Shigella naturally possess a Tol-Pal system which is made up of TolA, TolB, TolQ, TolR and Pal proteins. According to the invention, the natural Tol-Pal system is disrupted, thereby causing the bacterium to release greater quantities of outer membrane blebs into its culture medium during bacterial replication. Various disruptions can be made.

In some embodiments, at least one of the five Tol-Pal proteins is removed e.g. by deletion or inactivation of the gene encoding the protein. Thus the bacterium may express 0, 1, 2, 3 or 4 of TolA, TolB, TolQ, TolR and Pal proteins. Removal of one of the five proteins can suffice, in which case the bacterium expresses only 4 of these proteins. Preferably the TolR protein is removed e.g. by inactivation of a starting strain's tolR gene. Thus the bacterium may be tolA⁺ tolB⁺ tolQ⁺ TolR⁻ Pat⁺.

In some embodiments, the bacterium expresses all five Tol-Pal proteins, but at least one is mutated to cause hyperblebbing. For instance, the TolA, TolQ, TolR and/or Pal protein may be mutated such that the protein retains its membrane localisation but its interactions with other members of the Tol-Pal system are disrupted. The bacterium will thus retain TolA, TolQ and TolR as transmembrane proteins in the inner membrane, and Pal protein as a periplasm-facing lipoprotein in the outer membrane, but at least one of the TolA, TolQ, TolR and/or Pal proteins is mutated.

Examples of wild-type Shigella amino acid sequences of the TolA, TolB, TolQ, TolR and Pal proteins are given in the sequence listing as SEQ ID NOs: 1 to 5.

The Shigella Bacterium

The invention can be used with any of serogroups S. dysenteriae, S. flexneri, S. boydii and S. sonnies.

In addition to having a disrupted Tol-Pal system, thereby causing the bacterium to release greater quantities of outer membrane blebs into its culture medium during bacterial replication, a Shigella of the invention can advantageously include one or more further changes relative to a wild-type strain. These changes can be used in particular to remove components from the bacterium which would be toxic or undesirable in a human vaccine.

For example, a bacterium may not express native Shigella lipopolysaccharide (LPS), thereby reducing endotoxic activity. Various modifications can be made to prevent synthesis of native LPS, and these may disrupt the native lipid A structure, the oligosaccharide core, or the outer O antigen. For example, reference 3 reports LPS mutants caused by inactivation of the rfe and galU genes, and reference 4 reports LPS mutants caused by inactivation of yihE, galE, galK, galM and galT. Similarly, reference 5 reports defective LPS due to mutations in rfc, rfaL, or galU. Reference 6 reports LPS mutants caused by inactivation of msbB1 and msbB2, reducing acylation in lipid A. As shown herein, another LPS mutant with reduced lipid A acylation can be generated by inactivation of htrB [7,8].

Absence of O antigen in the LPS is preferred, thereby avoiding serotype-specific responses. In S. sonnies the O antigen is absent when the virulence plasmid is removed (see below). The galU gene codes for uridine diphosphoglucose (UDP-glucose) pyrophosphorylase and its inactivation results in synthesis of LPS with no attached O antigen. Inactivation of galU is useful for providing a Shigella without uridine diphosphoglucose pyrophosphorylase activity. Inactivation of rfbF and/or rfbG genes can be used to provide a Shigella without rhamnosyl transferase activity. Inactivation of rfc can be used to provide a Shigella without O antigen polymerase activity. Inactivation of all three of rfbF, rfbG and rfc can provide a useful strain.

Absence of hexa-acylated lipid A in the LPS is preferred. Loss of the virulence plasmid (see below) automatically leads to loss of the msbB2 gene, and the chromosomal msbB1 gene can be inactivated, thereby removing myristoyl transferase activity and providing a penta-acylated lipid A in the LPS.

Inactivation of the HtrB lauroyl transferase can provide Shigella with mainly tetra-acylated lipid A. Preferred strains have penta- or tetra-acylated LPS.

Preferred strains are inactivated for both galU and msbB1 and also lack the virulence plasmid, thereby providing a strain whose LPS is penta-acylated and lacks attached O antigen.

Some useful strains have penta- or tetra-acylated LPS which includes attached O antigen. More generally, though, preferred strains have penta- or tetra-acylated LPS which lacks attached O antigen. A S. flexneri strain with tolR, rfbG and htrB knockouts (and, optionally, rfbF and/or rfc inactivation) is useful. A useful S. sonnies strain has a tolR mutation and lacks a virulence plasmid.

A bacterium may not express an enteric toxin. For instance, a S. flexneri strain (particularly a 2a strain) may not express all of the subunits of Shigella enterotoxin 1 (ShET-1) e.g. the set1A and/or set1B genes can be inactivated. A S. dysenteriae strain may not express both subunits of Shiga toxin e.g. one or both of the stxA and/or stxB genes can be inactivated. A Shigella, particularly a S. sonnies or S. flexneri, may not express enterotoxin 2 (ShET-2) e.g. the ospD3 gene may be inactivated, or the virulence plasmid may be absent. Preferred strains encode none of ShET-1, ShET-2 and Shiga toxin.

Shigella bacteria of the invention can be prepared conveniently from wild-type or other starting strains using conventional techniques of mutagenesis e.g. see references 9 to 11. The lambda red recombination system is particularly useful with Shigella. Inactivation of a gene can be achieved in various ways e.g. by deletion or mutation in its promoter, by deletion or mutation of its start codon, by introduction of a premature stop codon, by deletion of the complete coding region, by knockout, etc. Isogenic knockout mutants are preferred. In the resulting Shigella bacterium, mRNA encoding the desired gene is absent and/or its translation is inhibited (e.g. to less than 1% of wild-type levels).

A Shigella bacterium of the invention may contain a marker gene in place of the inactivated gene e.g an antibiotic resistance marker. This can be achieved using homologous recombination. Preferably, though, unmarked deletions (i.e. deletion without introduction of a marker gene) are used.

Virulent Shigella strains possess a 220 kb plasmid that mediates virulence properties. This “virulence plasmid” has been shown to encode the genes for several aspects of Shigella virulence, including adhesins for target epithelial cells, the invasion plasmid antigens, virF, virG, etc. A Shigella of the invention may possess a virulence plasmid but, preferably, it does not possess a virulence plasmid. Absence of the plasmid can stabilise the strain during industrial culture, attenuate the strain by removing virulence factors (thereby increasing safety of manufacture), disrupt the lipopolysaccharide (the biosynthesis genes for the O antigen are on the plasmid in S. sonnies), avoid the presence of the ShET-2 enterotoxin (encoded by the ospD3 or sen gene on the plasmid), and avoid the presence of msbB2 which is a second copy of the msbB gene responsible for acylation of lipid A.

A Shigella of the invention may express one or more heterologous proteins e.g. proteins which are not naturally found in Shigella. If the heterologous protein is an outer membrane protein then blebs from the strain can be used as a delivery system for presenting non-Shigella antigens to the immune system.

Culture conditions for growing Shigella are well known in the art e.g. see references 12 to 14. For example, they may be grown using an organic nitrogen source (such as amino acid mixtures e.g. containing Ala, Arg, Asn, Asp; casamino acids may be used), glycerol as a carbon source, etc. Inclusion of L-aspartic acid in the medium is particularly useful and may function as both a nitrogen and carbon source.

Advantageously, Shigella of the invention may be grown under iron-limiting conditions as this has been shown to up-regulate iron-regulated proteins which are immunogenic and highly-conserved among Shigella spp. For instance, the bacteria may be grown in the presence of a compound such as desferal or 2,2′-dipyridyl or 8-hydroxyquinoline. Under these conditions the bacterium may increase expression of proteins such as the FepA outer membrane receptor, the colicin I receptor (CirA), and/or the ferric siderophore receptor (FhuA).

Blebs and Hyperblebbing Shigella bacteria of the invention are, relative to their corresponding wild-type strains, hyperblebbing i.e. they release into their culture medium larger quantities of blebs than the wild-type strain. These blebs are useful as components of Shigella vaccines.

The blebs typically have a diameter of 35-120 nm by electron microscopy e.g. 50 nm diameter.

The blebs are released spontaneously during bacterial growth and can be purified from the culture medium. The purification ideally involves separating the blebs from living and/or intact Shigella bacteria e.g. by size-based filtration using a filter, such as a 0.22 mm filter, which allows the blebs to pass through but which does not allow intact bacteria to pass through, or by using low speed centrifugation to pellet cells while leaving blebs in suspension.

Thus, unlike the culture medium, bleb-containing compositions of the invention will generally be substantially free from whole bacteria, whether living or dead. The size of the blebs means that they can readily be separated from whole bacteria by filtration e.g. as typically used for filter sterilisation. Although blebs will pass through a standard 0.22 mm filters, these can rapidly become clogged by other material, and so it may be useful to perform sequential steps of filter sterilisation through a series of filters of decreasing pore size before using a 0.22 μm filter. Examples of preceding filters would be those with pore size of 0.8 μm, 0.45 μm, etc.

Separation of spontaneously-released blebs from the culture medium is more convenient than methods which involve deliberate disruption of the outer membrane (e.g. by detergent treatment or sonication) to cause outer membrane vesicles to form. Moreover, they are substantially free from inner membrane and cytoplasmic contamination.

Blebs of the invention contain lipids and proteins. The protein content of the blebs has been analysed, and they include the proteins listed in Table 1 and discussed below.

Shigella Outer Membrane Proteins

Table 1 lists the GenBank name and GI number for 129 proteins which were detected in Shigella blebs of the invention. These 127 proteins may be used as immunogenic components in purified form, separate from blebs. A preferred subset of the 129 is the first 60 in the list (“Subset 3”).

Polypeptides can be prepared by various means e.g. by chemical synthesis (at least in part), by digesting longer polypeptides using proteases, by translation from RNA, by purification from cell culture (e.g. from recombinant expression or from Shigella culture). etc. Heterologous expression in an E. coli host is a preferred expression route.

Polypeptides of the invention may be attached or immobilised to a solid support. Polypeptides of the invention may comprise a detectable label e.g. a radioactive label, a fluorescent label, or a biotin label. This is particularly useful in immunoassay techniques.

Polypeptides can take various forms (e.g. native, fusions, glycosylated, non-glycosylated, lipidated, disulfide bridges, etc.). Polypeptides are preferably Shigella polypeptides.

Polypeptides are preferably prepared in substantially pure or substantially isolated form (i.e. substantially free from other Shigella or host cell polypeptides) or substantially isolated form. In general, the polypeptides are provided in a non-naturally occurring environment e.g. they are separated from their naturally-occurring environment. In certain embodiments, the subject polypeptide is present in a composition that is enriched for the polypeptide as compared to a control. As such, purified polypeptide is provided, whereby purified is meant that the polypeptide is present in a composition that is substantially free of other expressed polypeptides, where by substantially free is meant that less than 50%, usually less than 30% and more usually less than 10% of the composition is made up of other expressed polypeptides.

The term “polypeptide” refers to amino acid polymers of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. Polypeptides can occur as single chains or associated chains.

Pharmaceutical Compositions

The invention provides a pharmaceutical composition comprising (a) blebs of the invention and (b) a pharmaceutically acceptable carrier. The invention also provides a process for preparing such a composition, comprising the step of admixing blebs of the invention with a pharmaceutically acceptable carrier.

The invention also provides a pharmaceutical composition comprising (a) the bleb-free immunogenic composition defined above and (b) a pharmaceutically acceptable carrier.

The immunogenic composition may include a pharmaceutically acceptable carrier, which can be any substance that does not itself induce the production of antibodies harmful to the patient receiving the composition, and which can be administered without undue toxicity. Pharmaceutically acceptable carriers can include liquids such as water, saline, glycerol and ethanol. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, can also be present in such vehicles. A thorough discussion of suitable carriers is available in ref. 15.

Shigella infections can affect various areas of the body and so the compositions of the invention may be prepared in various forms. For example, the compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The composition may be prepared for topical administration e.g. as an ointment, cream or powder. The composition be prepared for oral administration e.g. as a tablet or capsule, or as a syrup (optionally flavoured). The composition may be prepared for pulmonary administration e.g. as an inhaler, using a fine powder or a spray. The composition may be prepared as a suppository or pessary. The composition may be prepared for nasal, aural or ocular administration e.g. as drops.

The composition is preferably sterile. It is preferably pyrogen-free. It is preferably buffered e.g. at between pH 6 and pH 8, generally around pH 7. Compositions of the invention may be isotonic with respect to humans.

Immunogenic compositions comprise an immunologically effective amount of immunogen, as well as any other of other specified components, as needed. By ‘immunologically effective amount’, it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

Previous work with vesicle vaccines (e.g. for meningococcus) offers pharmaceutical, posological and formulation guidance for administering blebs. The concentration of blebs in compositions of the invention will generally be between 10 and 500 μg/ml, preferably between 25 and 200 μg/ml, and more preferably about 50 μg/ml or about 100 μg/ml (expressed in terms of total protein in the blebs). A dosage volume of 0.5 ml is typical for injection.

The composition may be administered in conjunction with other immunoregulatory agents.

Adjuvants which may be used in compositions of the invention (particularly in bleb-free compositions) include, but are not limited to:

A. Mineral-Containing Compositions

Mineral containing compositions suitable for use as adjuvants in the invention include mineral salts, such as aluminium salts and calcium salts. The invention includes mineral salts such as hydroxides (e.g. oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates), sulphates, etc. [e.g. see chapters 8 & 9 of ref. 19], or mixtures of different mineral compounds, with the compounds taking any suitable form (e.g. gel, crystalline, amorphous, etc.), and with adsorption being preferred. The mineral containing compositions may also be formulated as a particle of metal salt.

The adjuvants known as “aluminium hydroxide” are typically aluminium oxyhydroxide salts, which are usually at least partially crystalline. Aluminium oxyhydroxide, which can be represented by the formula AlO (OH), can be distinguished from other aluminium compounds, such as aluminium hydroxide Al(OH)₃, by infrared (IR) spectroscopy, in particular by the presence of an adsorption band at 1070 cm⁻¹ and a strong shoulder at 3090-3100 cm⁻¹ [chapter 9 of ref. 19]. The degree of crystallinity of an aluminium hydroxide adjuvant is reflected by the width of the diffraction band at half height (WHH), with poorly-crystalline particles showing greater line broadening due to smaller crystallite sizes. The surface area increases as WHH increases, and adjuvants with higher WHH values have been seen to have greater capacity for antigen adsorption. A fibrous morphology (e.g. as seen in transmission electron micrographs) is typical for aluminium hydroxide adjuvants. The pI of aluminium hydroxide adjuvants is typically about 11 i.e. the adjuvant itself has a positive surface charge at physiological pH. Adsorptive capacities of between 1.8-2.6 mg protein per mg Al⁺⁺⁺ at pH 7.4 have been reported for aluminium hydroxide adjuvants.

The adjuvants known as “aluminium phosphate” are typically aluminium hydroxyphosphates, often also containing a small amount of sulfate (i.e. aluminium hydroxyphosphate sulfate). They may be obtained by precipitation, and the reaction conditions and concentrations during precipitation influence the degree of substitution of phosphate for hydroxyl in the salt. Hydroxyphosphates generally have a PO₄/Al molar ratio between 0.3 and 1.2. Hydroxyphosphates can be distinguished from strict AlPO₄ by the presence of hydroxyl groups. For example, an IR spectrum band at 3164 cm⁻¹ (e.g. at 200° C.) indicates the presence of structural hydroxyls [ch. 9 of ref. 19].

The PO₄/Al³⁺ molar ratio of an aluminium phosphate adjuvant will generally be between 0.3 and 1.2, preferably between 0.8 and 1.2, and more preferably 0.95+0.1. The aluminium phosphate will generally be amorphous, particularly for hydroxyphosphate salts. A typical adjuvant is amorphous aluminium hydroxyphosphate with PO₄/Al molar ratio between 0.84 and 0.92, included at 0.6 mg Al³⁺/ml. The aluminium phosphate will generally be particulate (e.g. plate-like morphology as seen in transmission electron micrographs). Typical diameters of the particles are in the range 0.5-20 μm (e.g. about 5-10 μm) after any antigen adsorption. Adsorptive capacities of between 0.7-1.5 mg protein per mg Al⁺⁺⁺ at pH 7.4 have been reported for aluminium phosphate adjuvants.

The point of zero charge (PZC) of aluminium phosphate is inversely related to the degree of substitution of phosphate for hydroxyl, and this degree of substitution can vary depending on reaction conditions and concentration of reactants used for preparing the salt by precipitation. PZC is also altered by changing the concentration of free phosphate ions in solution (more phosphate=more acidic PZC) or by adding a buffer such as a histidine buffer (makes PZC more basic). Aluminium phosphates used according to the invention will generally have a PZC of between 4.0 and 7.0, more preferably between 5.0 and 6.5 e.g. about 5.7.

Suspensions of aluminium salts used to prepare compositions of the invention may contain a buffer (e.g. a phosphate or a histidine or a Tris buffer), but this is not always necessary. The suspensions are preferably sterile and pyrogen-free. A suspension may include free aqueous phosphate ions e.g.

present at a concentration between 1.0 and 20 mM, preferably between 5 and 15 mM, and more preferably about 10 mM. The suspensions may also comprise sodium chloride.

In one embodiment, an adjuvant component includes a mixture of both an aluminium hydroxide and an aluminium phosphate. In this case there may be more aluminium phosphate than hydroxide e.g. a weight ratio of at least 2:1 e.g. >5:1, >6:1, >7:1, >8:1, >9:1, etc.

The concentration of Al⁺⁺⁺ in a composition for administration to a patient is preferably less than 10 mg/ml e.g. <5 mg/ml, <4 mg/ml, <3 mg/ml, <2 mg/ml, <1 mg/ml, etc. A preferred range is between 0.3 and 1 mg/ml. A maximum of <0.85mg/dose is preferred.

B. Oil Emulsions Oil emulsion compositions suitable for use as adjuvants in the invention include squalene-water emulsions, such as MF59 [Chapter 10 of ref. 19; see also ref. 16] (5% Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into submicron particles using a microfluidizer). Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) may also be used.

Various suitable oin-in-water emulsions are known, and they typically include at least one oil and at least one surfactant, with the oil(s) and surfactant(s) being biodegradable (metabolisable) and biocompatible. The oil droplets in the emulsion are generally less than 5 μm in diameter, and advantageously the emulsion comprises oil droplets with a sub-micron diameter, with these small sizes being achieved with a microfluidiser to provide stable emulsions. Droplets with a size less than 220 nm are preferred as they can be subjected to filter sterilization.

The invention can be used with oils such as those from an animal (such as fish) or vegetable source. Sources for vegetable oils include nuts, seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil, the most commonly available, exemplify the nut oils. Jojoba oil can be used e.g. obtained from the jojoba bean. Seed oils include safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil and the like. In the grain group, corn oil is the most readily available, but the oil of other cereal grains such as wheat, oats, rye, rice, teff, triticale and the like may also be used. 6-10 carbon fatty acid esters of glycerol and 1,2-propanediol, while not occurring naturally in seed oils, may be prepared by hydrolysis, separation and esterification of the appropriate materials starting from the nut and seed oils. Fats and oils from mammalian milk are metabolizable and may therefore be used in the practice of this invention. The procedures for separation, purification, saponification and other means necessary for obtaining pure oils from animal sources are well known in the art. Most fish contain metabolizable oils which may be readily recovered. For example, cod liver oil, shark liver oils, and whale oil such as spermaceti exemplify several of the fish oils which may be used herein. A number of branched chain oils are synthesized biochemically in 5-carbon isoprene units and are generally referred to as terpenoids. Shark liver oil contains a branched, unsaturated terpenoid known as squalene, 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene. Other preferred oils are the tocopherols (see below). Oil in water emulsions comprising sqlauene are particularly preferred. Mixtures of oils can be used.

Surfactants can be classified by their ‘HLB’ (hydrophile/lipophile balance). Preferred surfactants of the invention have a HLB of at least 10, preferably at least 15, and more preferably at least 16. The invention can be used with surfactants including, but not limited to: the polyoxyethylene sorbitan esters surfactants (commonly referred to as the Tweens), especially polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO), sold under the DOWFAX™ tradename, such as linear EO/PO block copolymers; octoxynols, which can vary in the number of repeating ethoxy (oxy-1,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being of particular interest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipids such as phosphatidylcholine (lecithin); polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethyleneglycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as the SPANs), such as sorbitan trioleate (Span 85) and sorbitan monolaurate. Preferred surfactants for including in the emulsion are Tween 80 (polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate), lecithin and Triton X-100. As mentioned above, detergents such as Tween 80 may contribute to the thermal stability seen in the examples below. Mixtures of surfactants can be used e.g. Tween 80/Span 85 mixtures. A combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (Tween 80) and an octoxynol such as t-octylphenoxypolyethoxyethanol (Triton X-100) is also suitable. Another useful combination comprises laureth 9 plus a polyoxyethylene sorbitan ester and/or an octoxynol.

Preferred amounts of surfactants (% by weight) are: polyoxyethylene sorbitan esters (such as Tween 80) 0.01 to 1%, in particular about 0.1%; octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100, or other detergents in the Triton series) 0.001 to 0.1%, in particular 0.005 to 0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 20%, preferably 0.1 to 10% and in particular 0.1 to 1% or about 0.5%.

Specific oil-in-water emulsion adjuvants useful with the invention include, but are not limited to:

-   -   A submicron emulsion of squalene, Tween 80, and Span 85. The         composition of the emulsion by volume can be about 5% squalene,         about 0.5% polysorbate 80 and about 0.5% Span 85. In weight         terms, these ratios become 4.3% squalene, 0.5% polysorbate 80         and 0.48% Span 85. This adjuvant is known as ‘MF59’ [16-18], as         described in more detail in Chapter 10 of ref. 19 and chapter 12         of ref 20. The MF59 emulsion advantageously includes citrate         ions e.g. 10 mM sodium citrate buffer.     -   An emulsion comprising squalene, an α-tocopherol, and         polysorbate 80. These emulsions may have from 2 to 10% squalene,         from 2 to 10% tocopherol and from 0.3 to 3% Tween 80, and the         weight ratio of squalene:tocopherol is preferably ≦1 (e.g. 0.90)         as this provides a more stable emulsion. Squalene and Tween 80         may be present volume ratio of about 5:2, or at a weight ratio         of about 11:5. One such emulsion can be made by dissolving Tween         80 in PBS to give a 2% solution, then mixing 90 ml of this         solution with a mixture of (5 g of DL-α-tocopherol and 5 ml         squalene), then microfluidising the mixture. The resulting         emulsion may have submicron oil droplets e.g. with an average         diameter of between 100 and 250 nm, preferably about 180 nm.     -   An emulsion of squalene, a tocopherol, and a Triton detergent         (e.g. Triton X-100). The emulsion may also include a 3d-MPL (see         below). The emulsion may contain a phosphate buffer.     -   An emulsion comprising a polysorbate (e.g. polysorbate 80), a         Triton detergent (e.g. Triton X-100) and a tocopherol (e.g. an         α-tocopherol succinate). The emulsion may include these three         components at a mass ratio of about 75:11:10 (e.g. 750 μg/ml         polysorbate 80, 110 μg/ml Triton X-100 and 100 μg/ml         α-tocopherol succinate), and these concentrations should include         any contribution of these components from antigens. The emulsion         may also include squalene. The emulsion may also include a         3d-MPL (see below). The aqueous phase may contain a phosphate         buffer.     -   An emulsion of squalane, polysorbate 80 and poloxamer 401         (“Pluronic™ L121”). The emulsion can be formulated in phosphate         buffered saline, pH 7.4. This emulsion is a useful delivery         vehicle for muramyl dipeptides, and has been used with         threonyl-MDP in the “SAF-1” adjuvant [21] (0.05-1% Thr-MDP, 5%         squalane, 2.5% Pluronic L121 and 0.2% polysorbate 80). It can         also be used without the Thr-MDP, as in the “AF” adjuvant [22]         (5% squalane, 1.25% Pluronic L121 and 0.2% polysorbate 80).         Microfluidisation is preferred.     -   An emulsion comprising squalene, an aqueous solvent, a         polyoxyethylene alkyl ether hydrophilic nonionic surfactant         (e.g. polyoxyethylene (12) cetostearyl ether) and a hydrophobic         nonionic surfactant (e.g. a sorbitan ester or mannide ester,         such as sorbitan monoleate or ‘Span 80’). The emulsion is         preferably thermoreversible and/or has at least 90% of the oil         droplets (by volume) with a size less than 200 nm [23]. The         emulsion may also include one or more of: alditol; a         cryoprotective agent (e.g. a sugar, such as dodecylmaltoside         and/or sucrose); and/or an alkylpolyglycoside. Such emulsions         may be lyophilized.     -   An emulsion having from 0.5-50% of an oil, 0.1-10% of a         phospholipid, and 0.05-5% of a non-ionic surfactant. As         described in reference 24, preferred phospholipid components are         phosphatidylcholine, phosphatidylethanolamine,         phosphatidylserine, phosphatidylinositol, phosphatidylglycerol,         phosphatidic acid, sphingomyelin and cardiolipin. Submicron         droplet sizes are advantageous.     -   A submicron oil-in-water emulsion of a non-metabolisable oil         (such as light mineral oil) and at least one surfactant (such as         lecithin, Tween 80 or Span 80). Additives may be included, such         as QuilA saponin, cholesterol, a saponin-lipophile conjugate         (such as GPI-0100, described in reference 25, produced by         addition of aliphatic amine to desacylsaponin via the carboxyl         group of glucuronic acid), dimethyidioctadecylammonium bromide         and/or N,N-dioctadecyl-N,N-bis (2-hydroxyethyl)propanediamine.     -   An emulsion comprising a mineral oil, a non-ionic lipophilic         ethoxylated fatty alcohol, and a non-ionic hydrophilic         surfactant (e.g. an ethoxylated fatty alcohol and/or         polyoxyethylene-polyoxypropylene block copolymer) [26].     -   An emulsion comprising a mineral oil, a non-ionic hydrophilic         ethoxylated fatty alcohol, and a non-ionic lipophilic surfactant         (e.g. an ethoxylated fatty alcohol and/or         polyoxyethylene-polyoxypropylene block copolymer) [26].     -   An emulsion in which a saponin (e.g. QuilA or QS21) and a sterol         (e.g. a cholesterol) are associated as helical micelles [27].

Antigens and adjuvants in a composition will typically be in admixture at the time of delivery to a patient. The emulsions may be mixed with antigen during manufacture, or extemporaneously, at the time of delivery. Thus the adjuvant and antigen may be kept separately in a packaged or distributed vaccine, ready for final formulation at the time of use. The antigen will generally be in an aqueous form, such that the vaccine is finally prepared by mixing two liquids. The volume ratio of the two liquids for mixing can vary (e.g. between 5:1 and 1:5) but is generally about 1:1.

C. Saponin Formulations [Chapter 22 of Ref 19]

Saponin formulations may also be used as adjuvants in the invention. Saponins are a heterogeneous group of sterol glycosides and triterpenoid glycosides that are found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. Saponin from the bark of the Quillaia saponaria Molina tree have been widely studied as adjuvants. Saponin can also be commercially obtained from Smilax ornata (sarsaprilla), Gypsophilla paniculata (brides veil), and Saponaria officianalis (soap root). Saponin adjuvant formulations include purified formulations, such as QS21, as well as lipid formulations, such as ISCOMs. QS21 is marketed as Stimulon™.

Saponin compositions have been purified using HPLC and RP-HPLC. Specific purified fractions using these techniques have been identified, including QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C. Preferably, the saponin is QS21. A method of production of QS21 is disclosed in ref. 28. Saponin formulations may also comprise a sterol, such as cholesterol [29].

Combinations of saponins and cholesterols can be used to form unique particles called immunostimulating complexs (ISCOMs; see chapter 23 of ref. 19; also refs 30 & 31). ISCOMs typically also include a phospholipid such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in ISCOMs. Preferably, the ISCOM includes one or more of QuilA, QHA & QHC. Optionally, the ISCOMS may be devoid of additional detergent [32].

A review of the development of saponin based adjuvants can be found in refs. 33 & 34.

D. Bacterial or Microbial Derivatives

Adjuvants suitable for use in the invention include bacterial or microbial derivatives such as non-toxic derivatives of enterobacterial lipopolysaccharide (LPS), Lipid A derivatives, immunostimulatory oligonucleotides and ADP-ribosylating toxins and detoxified derivatives thereof.

Non-toxic derivatives of LPS include monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3 de-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred “small particle” form of 3 De-O-acylated monophosphoryl lipid A is disclosed in ref. 35. Such “small particles” of 3dMPL are small enough to be sterile filtered through a 0.22 μm membrane [35]. Other non-toxic LPS derivatives include monophosphoryl lipid A mimics, such as aminoalkyl glucosaminide phosphate derivatives e.g. RC-529 [36,37].

Lipid A derivatives include derivatives of lipid A from Escherichia coli such as OM-174. OM-174 is described for example in refs. 38 & 39.

Immunostimulatory oligonucleotides suitable for use as adjuvants in the invention include nucleotide sequences containing a CpG motif (a dinucleotide sequence containing an unmethylated cytosine linked by a phosphate bond to a guanosine). Double-stranded RNAs and oligonucleotides containing palindromic or poly(dG) sequences have also been shown to be immunostimulatory.

The CpG's can include nucleotide modifications/analogs such as phosphorothioate modifications and can be double-stranded or single-stranded. References 40, 41 and 42 disclose possible analog substitutions e.g. replacement of guanosine with 2′-deoxy-7-deazaguanosine. The adjuvant effect of CpG oligonucleotides is further discussed in refs. 43-48.

The CpG sequence may be directed to TLR9, such as the motif GTCGTT or TTCGTT [49]. The CpG sequence may be specific for inducing a Th1 immune response, such as a CpG-A ODN, or it may be more specific for inducing a B cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs are discussed in refs. 50-52. Preferably, the CpG is a CpG-A ODN.

Preferably, the CpG oligonucleotide is constructed so that the 5′ end is accessible for receptor recognition. Optionally, two CpG oligonucleotide sequences may be attached at their 3′ ends to form “inununomers”. See, for example, refs. 53-55.

A particularly useful adjuvant based around immunostimulatory oligonucleotides is known as IC-31™ [56-58]. Thus an adjuvant used with the invention may comprise a mixture of (i) an oligonucleotide (e.g. between 15-40 nucleotides) including at least one (and preferably multiple) CpI motifs (i.e. a cytosine linked to an inosine to form a dinucleotide), and (ii) a polycationic polymer, such as an oligopeptide (e.g. between 5-20 amino acids) including at least one (and preferably multiple) Lys-Arg-Lys tripeptide sequence(s). The oligonucleotide may be a deoxynucleotide comprising 26-mer sequence 5′-(IC)₁₃-3′ (SEQ ID NO: 7). The polycationic polymer may be a peptide comprising 11-mer amino acid sequence KLKLLLLLKLK (SEQ ID NO: 6). This combination of SEQ ID NOs: 6 and 7 provides the IC-31™ adjuvant.

Bacterial ADP-ribosylating toxins and detoxified derivatives thereof may be used as adjuvants in the invention. Preferably, the protein is derived from E. coli (E. coli heat labile enterotoxin “LT”), cholera (“CT”), or pertussis (“PT”). The use of detoxified ADP-ribosylating toxins as mucosal adjuvants is described in ref. 59 and as parenteral adjuvants in ref. 60. The toxin or toxoid is preferably in the form of a holotoxin, comprising both A and B subunits. Preferably, the A subunit contains a detoxifying mutation; preferably the B subunit is not mutated. Preferably, the adjuvant is a detoxified LT mutant such as LT-K63, LT-R72, and LT-G192. The use of ADP-ribosylating toxins and detoxified derivatives thereof, particularly LT-K63 and LT-R72, as adjuvants can be found in refs. 61-68. A useful CT mutant is or CT-E29H [69]. Numerical reference for amino acid substitutions is preferably based on the alignments of the A and B subunits of ADP-ribosylating toxins set forth in ref. 70, specifically incorporated herein by reference in its entirety.

E. Human Immunomodulators

Human immunomodulators suitable for use as adjuvants in the invention include cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 [71], etc.) [72], interferons (e.g. interferon-y), macrophage colony stimulating factor, and tumor necrosis factor. A preferred immunomodulator is IL-12.

F. Bioadhesives and Mucoadhesives

Bioadhesives and mucoadhesives may also be used as adjuvants in the invention. Suitable bioadhesives include esterified hyaluronic acid microspheres [73] or mucoadhesives such as cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides and carboxymethylcellulose. Chitosan and derivatives thereof may also be used as adjuvants in the invention [74].

G. Microparticles

Microparticles may also be used as adjuvants in the invention. Microparticles (i.e. a particle of ˜A 100 nm to ˜150 μm in diameter, more preferably ˜200 nm to ˜30 μm in diameter, and most preferably ˜500 nm to ˜10 μm in diameter) formed from materials that are biodegradable and non-toxic (e.g. a poly(α-hydroxy acid), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.), with poly(lactide-co-glycolide) are preferred, optionally treated to have a negatively-charged surface (e.g. with SDS) or a positively-charged surface (e.g. with a cationic detergent, such as CTAB).

H. Liposomes (Chapters 13 & 14 of Ref. 19)

Examples of liposome formulations suitable for use as adjuvants are described in refs. 75-77.

I. Inidazoquinolone Compounds.

Examples of imidazoquinolone compounds suitable for use adjuvants in the invention include

Imiquamod and its homologues (e.g. “Resiquimod 3M”), described further in refs. 78 and 79.

The invention may also comprise combinations of aspects of one or more of the adjuvants identified above. For example, the following adjuvant compositions may be used in the invention: (1) a saponin and an oil-in-water emulsion [80]; (2) a saponin (e.g. QS21)+a non-toxic LPS derivative (e.g. 3dMPL) [81]; (3) a saponin (e.g. QS21)+a non-toxic LPS derivative (e.g. 3dMPL)+a cholesterol; (4) a saponin (e.g. QS21)+3dMPL+IL-12 (optionally+a sterol) [82]; (5) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions [83]; (6) SAF, containing 10% squalane, 0.4% Tween 80™, 5% pluronic-block polymer L121, and thr-MDP, either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion. (7) Ribi™ adjuvant system (RAS), (Ribi Immunochem) containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox™); and (8) one or more mineral salts (such as an aluminum salt)+a non-toxic derivative of LPS (such as 3dMPL).

Other substances that act as immunostimulating agents are disclosed in chapter 7 of ref. 19.

An aluminium hydroxide adjuvant is useful, and antigens are generally adsorbed to this salt. Oil-in-water emulsions comprising squalene, with submicron oil droplets, are also preferred, particularly in the elderly. Useful adjuvant combinations include combinations of Th1 and Th2 adjuvants such as CpG & an aluminium salt, or resiquimod & an aluminium salt. A combination of an aluminium salt and 3dMPL may be used.

Immunisation

In addition to providing immunogenic compositions as described above, the invention also provides a method for raising an antibody response in a mammal, comprising administering an immunogenic composition of the invention to the mammal. The antibody response is preferably a protective antibody response. The invention also provides compositions of the invention for use in such methods.

The invention also provides a method for protecting a mammal against a Shigella infection and/or disease (e.g. against shigellosis, Reiter's syndrome, and/or hemolytic uremic syndrome), comprising administering to the mammal an immunogenic composition of the invention.

The invention provides compositions of the invention for use as medicaments (e.g. as immunogenic compositions or as vaccines). It also provides the use of vesicles of the invention in the manufacture of a medicament for preventing a Shigella infection in a mammal e.g. for preventing shigellosis, Reiter's syndrome, and/or hemolytic uremic syndrome. It also provides the use of a bleb protein (as defined above) in the manufacture of a bleb-free medicament for preventing a Shigella infection in a mammal e.g. for preventing shigellosis.

The mammal is preferably a human. The human may be an adult or, preferably, a child. Where the vaccine is for prophylactic use, the human is preferably a child (e.g. a toddler or infant); where the vaccine is for therapeutic use, the human is preferably an adult. A vaccine intended for children may also be administered to adults e.g. to assess safety, dosage, immunogenicity, etc.

The uses and methods are particularly useful for preventing/treating diseases including, but not limited to, shigellosis, Reiter's syndrome, and/or hemolytic uremic syndrome

Efficacy of therapeutic treatment can be tested by monitoring Shigella infection after administration of the composition of the invention. Efficacy of prophylactic treatment can be tested by monitoring immune responses against immunogenic proteins in the blebs or other antigens after administration of the composition. Immunogenicity of compositions of the invention can be determined by administering them to test subjects (e.g. children 12-16 months age) and then determining standard serological parameters. These immune responses will generally be determined around 4 weeks after administration of the composition, and compared to values determined before administration of the composition. Where more than one dose of the composition is administered, more than one post-administration determination may be made.

Compositions of the invention will generally be administered directly to a patient. Direct delivery may be accomplished by parenteral injection (e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue), or by rectal, oral, vaginal, topical, transdermal, intranasal, ocular, aural, pulmonary or other mucosal administration. Intramuscular administration to the thigh or the upper arm is preferred. Injection may be via a needle (e.g. a hypodermic needle), but needle-free injection may alternatively be used. A typical intramuscular dose is about 0.5 ml.

The invention may be used to elicit systemic and/or mucosal immunity.

Dosage treatment can be a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunisation schedule and/or in a booster immunisation schedule. A primary dose schedule may be followed by a booster dose schedule. Suitable timing between priming doses (e.g. between 4-16 weeks), and between priming and boosting, can be routinely determined.

General

The term “comprising” encompasses “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X+Y.

The term “about” in relation to a numerical value x is optional and means, for example, x±10%.

The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.

References to a percentage sequence identity between two amino acid sequences means that, when aligned, that percentage of amino acids are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in section 7.7.18 of reference 84. A preferred alignment is determined by the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. The Smith-Waterman homology search algorithm is well known and is disclosed in reference 85.

“GI” numbering is used above. A GI number, or “GenInfo Identifier”, is a series of digits assigned consecutively to each sequence record processed by NCBI when sequences are added to its databases. The GI number bears no resemblance to the accession number of the sequence record. When a sequence is updated (e.g. for correction, or to add more annotation or information) then it receives a new GI number. Thus the sequence associated with a given GI number is never changed.

Where the invention concerns an “epitope”, this epitope may be a B-cell epitope and/or a T-cell epitope. Such epitopes can be identified empirically (e.g. using PEPSCAN [86,87] or similar methods), or they can be predicted (e.g. using the Jameson-Wolf antigenic index [88], matrix-based approaches [89], MAPITOPE [90], TEPITOPE [91,92], neural networks [93], OptiMer & EpiMer [94, 95], ADEPT [96], Tsites [97], hydrophilicity [98], antigenic index [99] or the methods disclosed in references 100-101, etc.). Epitopes are the parts of an antigen that are recognised by and bind to the antigen binding sites of antibodies or T-cell receptors, and they may also be referred to as “antigenic determinants”.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an electron micrograph of blebs of the invention purified from culture.

FIG. 2 shows 2D SDS-PAGE of blebs from S. sonnies ΔtolR ΔgalU.

FIG. 3 shows LPS of blebs in various indicated strains, stained with anti-core antibody.

FIG. 4 shows an immunoblot of 2D-separated proteins of blebs using serum from immunised mice.

FIG. 5 shows SDS-PAGE of bacteria grown in different conditions.

FIG. 6 shows adsorbed proteins in blebs. Lanes from left to right: 1) protein molecular weight marker, 2) Ss OMB 10 μg, 3) Ss OMB 2 μg, 4) Ss OMB 10 μg adsorbed on alum for 1 month.

FIG. 7 shows FACS data for indicated strains.

MODES FOR CARRYING OUT THE INVENTION

Preparation of Mutant of S. sonnies

The tolR gene of wild-type S. sonnies 53G was deleted using the k Red system [11,102]. Competent cells transformed with the λ Red plasmids are prepared and then transformed with a linear fragment designed to swap the tolR gene for an antibiotic resistance gene by homologous recombination. Clones that have integrated the fragment into the chromosome are selected by resistance to the antibiotic and deletion of the tolR is verified by PCR or other techniques. The temperature sensitive λ Red plasmids can then be removed by growth of the new clones at 37° C.

The lack of TolR expression in this ΔtolR mutant was confirmed and, compared to the original wild-type isolate, it was confirmed to release more blebs into culture medium during growth.

The galU gene was also deleted in a similar way, to provide a ΔgalU single mutant and a ΔtolRΔgalU double mutant. Blebs released by mutants are confirmed to have a defective LPS lacking 0 antigen.

A ΔtolRΔmsbB double mutant strain with modified LPS is prepared in the same way.

The virulence plasmid has also been removed from the ΔtolR and ΔtolRΔmsbB strains.

Preparation of Mutant of S. flexneri

The tolR gene of S. flexneri was deleted using the λ Red system as described above for S. sonnies. O antigen biosynthesis in S. flexneri was abolished by deletion of a chromosomal fragment comprising the complete rfbG gene and as well as parts of dbF and rfc, resulting in activation of all three genes. The deletion was generated using the λ Red system and is abbreviated as ΔrfbG.

A ΔtolRΔrfbG double mutant has been generated in the same way.

A ΔtolRΔhtrB double mutant containing modified LPS has been generated in the same way.

The virulence plasmid has also been removed from these strains.

Purification of Blebs

Fermentation of the double mutant ΔtolRΔgalU strain was run under the following conditions: pH 7.1, 37° C., dissolved oxygen maintained at 30% saturation by controlling agitation and setting maximum aeration. The pH was controlled by addition of 4M ammonium hydroxide. The foam was controlled by addition of 10% PPG during the run. The medium consisted of the following components: KH₂PO₄ 5 g/l, K₂HPO₄ 20 g/l and yeast extract 30 g/l. After the medium was sterilized by autoclaving, glycerol 15 g/l and MgSO₄ 2 mM were added prior to inoculation. The culture inoculum was 5% of the fermentor volume. The fermentation process took approximately 13 hours and cell concentration was measured as optical density at 600 nm.

The fermentation process of the S. sonnies ΔtolRΔmsbB double mutant strain was performed with defined medium: glycerol 30 g/l, KH₂PO₄ 13.3 g/l, (NH₄)₂HPO₄ 4 g/l, MgSO₄·7H₂O (1M) 2 ml, citric acid 1.7 g/l, CoCl₂·6H₂O 2.5 mg/l, MnCl₂·4H₂O 15 mg/l, CuCl₂·2H₂O 1.5 mg/l, H₃BO₃ 3 mg/l, Na₂MoO₄·2H₂O 2.5 mg/l, Zn(CH₃COO)₂·2H₂O 13 mg/l, ferric citrate 2 μM, thiamine 50 mg/l, nicotinic acid 10 mg/l, L-acid aspartic 2.5 g/l.

Vesicles produced in the fermentation broth were purified using two consecutive TFF (tangential flow filtration) steps: micro-filtration at 0.22 μm and then a second micro-filtration at 0.1 μm. During the first filtration step the vesicles were separated from biomass by TFF through a 0.22 μm pore size cassette. The biomass was first concentrated 4-fold and, after five diafiltration steps against PBS, the vesicles were collected in the filtrate. In the second filtration step the filtrate from the 0.22 μm TFF was further micro-filtered trough a 0.1 μm cut-off cassette, in order to purify the vesicles from soluble proteins. The vesicles could not pass through the filter cassette. After five diafiltration steps, the retentate containing the vesicles was collected.

The final purified product was observed with TEM (FIG. 1). The blebs have a homogenous size of about 50 nm in diameter.

Blebs from S. flexneri mutants were purified in the same way after growing the various strains in yeast extract medium as used for S. sonnies

Bleb Characterisation

A proteomic approach confirmed that the blebs are essentially pure outer membranes. Unlike conventional outer membrane vesicles (OMV) derived by disruption of the outer membrane, the blebs conserve lipophilic proteins and are essentially free of cytoplasmic and inner membrane components.

Blebs from S. sonnies and S. flexneri strains were denatured with a detergent and proteins were identified with a LC-MS/MS approach. Alternatively, blebs were separated with SDS page or 2D gel electrophoresis (FIG. 2). Visible bands and spots were excised from the gel and proteins identified via protein mass fingerprint. The relative amount of different proteins was studied with densitometer analysis of SDS-PAGE bands or spots from the 2D gel.

FIG. 3 shows a shift in LPS mobility in the ΔgalU mutant strain compared to wildtype Shigella and E. coli (all strains are in a ΔtolR background).

A second proteomic approach, based on surface digestion, was used to characterize exposed portions of membrane proteins. A set of proteins was identified as reactive with sera from mice immunized with the blebs and many of these have been found to be conserved in a large panel of strains. Little is known about the structure of most integral outer membrane proteins. The surfome of blebs was investigated by treatment with a protease and recovery and identification via LC-MS/MS of released peptides. As blebs should represent the surface of the whole living bacterial cell, this map should be representative of exposed proteins on the surface of S. sonnies.

By these and other approaches the 129 proteins listed in Table 1 have been seen in the blebs.

Bleb Immunogenicity

Mice immunised with the blebs from the ΔtolRΔgalU strain produce serum which reacts with a 2D gel of the blebs as shown in FIG. 4. Thus the blebs are immunogenic.

Mice received 2 μg or 10 μg S. sonnies ΔtolRΔgalU blebs (measured as total protein), with or without adjuvant (aluminium hydroxide or Freund's complete). A classical ELISA method was performed to analyze IgG production in sera obtained from immunization studies. Sera from all groups of mice demonstrated a high level production of bleb-specific IgG. No significant differences in IgG production were detected when blebs were used alone or in combination with an adjuvant. Tthe group immunized with the lower dose of 2 μg showed the same level of bleb-specific IgG as the group immunized with 10 μg, showing that a low dose vaccine may be achievable i.e. more doses per dollar. Blebs from other S. sonnies as well as S. flexneri strains were similarly immunogenic.

Sera raised against the blebs were tested for reactivity with three different bacteria: S. sonnies G53, S. flexneri 2a 2457T or S. flexneri 5 M90T. The samples were than stained with a labeled secondary Ab and were analyzed by flow cytometry. As shown in FIG. 7, the S. sonnies and S. flexneri strains cross-react with the sera.

Therefore the bleb approach has a strong potential to produce effective and low-cost vaccines and can be extended to different Shigella strains towards a broad spectrum vaccine.

Bleb Adsorption

Blebs were combined with aluminium hydroxide (2 mg/ml) for adsorption. The adsorbed material was stored at 4° C. for 1 day, 1 week or 1 month. The blebs were toatlyl adsorbed after 1 day and there was no evidence of desorption even after 1 month (FIG. 6).

Iron-Limiting Growth

FIG. 5 shows proteins expressed by Shigella grown under various conditions. In lane 4 the bacteria were grown in the presence of 200 μM FeSO₄ whereas in lane 5 the culture had 200 μM dypiridyl. The inset shows that three proteins are up-regulated under the iron-limiting conditions. These three proteins were identified as the FepA outer membrane receptor (GI 74311118), the colicin I receptor (GI 74312677), and the putative ferric siderophore receptor (GI 74313972). These proteins are well-conserved among Shigella spp. and enterobacteriaceae and are potentially highly immunogenic. Thus growth of Shigella under iron-limiting conditions can lead to the release of blebs which are, compared to normal growth conditions, enriched for these proteins.

It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

TABLE 1 SEQ ID NO: GI Gene name Definition 8 56480244 tolC outer membrane channel protein [Shigella flexneri 2a str. 301] 9 74312736 ompC outer membrane porin protein C [Shigella sonnei Ss046] 10 74311514 ompA outer membrane protein A [Shigella sonnei Ss046] 11 110807342 SFV_3519 hypothetical protein SFV_3519 [Shigella flexneri 5 str. 8401] 12 56479734 ompX outer membrane protein X [Shigella flexneri 2a str. 301] 13 24113033 slyB putative outer membrane protein [Shigella flexneri 2a str. 301] 14 24112608 lolB outer membrane lipoprotein LolB [Shigella flexneri 2a str. 301] 15 24111612 yaeT outer membrane protein assembly factor YaeT [Shigella flexneri 2a str. 301] 16 187733369 outer membrane protein C [Shigella boydii CDC 3083-94] 17 24113066 Lpp murein lipoprotein [Shigella flexneri 2a str. 301] 18 56479690 pal peptidoglycan-associated outer membrane lipoprotein [Shigella flexneri 2a str. 301] 19 24115506 ecnB entericidin B membrane lipoprotein [Shigella flexneri 2a str. 301] 20 30063370 yedD hypothetical protein S2067 [Shigella flexneri 2a str. 2457T] 21 30064374 ygiW hypothetical protein S3269 [Shigella flexneri 2a str. 2457T] 22 30065519 yjel hypothetical protein S4565 [Shigella flexneri 2a str. 2457T] 23 24111837 ybaY hypothetical protein SF0398 [Shigella flexneri 2a str. 301] 24 24113773 SF2485 hypothetical protein SF2485 [Shigella flexneri 2a str. 301] 25 74313380 SSON_2966 hypothetical protein SSON_2966 [Shigella sonnei Ss046] 26 30063856 nlpB lipoprotein [Shigella flexneri 2a str. 2457T] 27 145294038 exc entry exclusion protein 2 [Shigella sonnei Ss046] 28 82775909 rlpB LPS-assembly lipoprotein RplB [Shigella dysenteriae Sd197] 29 74311310 ybhC putative pectinesterase [Shigella sonnei Ss046] 30 24114611 fkpA FKBP-type peptidyl-prolyl cis-trans isomerase [Shigella flexneri 2a str. 301] 31 74312826 hisJ histidine-binding periplasmic protein of high-affinity histidine transport system [Shigella sonnei Ss046] 32 24111599 htrA serine endoprotease [Shigella flexneri 2a str. 301] 33 30062097 tolB translocation protein TolB [Shigella flexneri 2a str. 2457T] 34 24111968 modA molybdate transporter periplasmic protein [Shigella flexneri 2a str. 301] 35 24114628 ppiA peptidyl-prolyl cis-trans isomerase A (rotamase A) [Shigella flexneri 2a str. 301] 36 24111499 surA peptidyl-prolyl cis-trans isomerase SurA [Shigella flexneri 2a str. 301] 37 30062764 oppA periplasmic oligopeptide binding protein [Shigella flexneri 2a str. 2457T] 38 30065614 osmY periplasmic protein [Shigella flexneri 2a str. 2457T] 39 74311404 artJ arginine 3rd transport system periplasmic binding protein [Shigella sonnei Ss046] 40 74311061 ushA bifunctional UDP-sugar hydrolase/5&apos;-nucleotidase periplasmic precursor [Shigella sonnei Ss046] 41 74311733 fliY cystine transporter subunit [Shigella sonnei Ss046] 42 110805056 mdoG glucan biosynthesis protein G [Shigella flexneri 5 str. 8401] 43 74312961 cysP thiosulfate transporter subunit [Shigella sonnei Ss046] 44 24114441 yraP hypothetical protein SF3191 [Shigella flexneri 2a str. 301] 45 74312191 SSON_1681 putative receptor [Shigella sonnei Ss046] 46 74312061 ydgA hypothetical protein SSON_1546 [Shigella sonnei Ss046] 47 24111764 proC pyrroline-5-carboxylate reductase [Shigella flexneri 2a str. 301] 48 24112431 SF1022 hypothetical protein SF1022 [Shigella flexneri 2a str. 301] 49 110806822 yggE hypothetical protein SFV_2968 [Shigella flexneri 5 str. 8401] 50 74312071 ydgH hypothetical protein SSON_1556 [Shigella sonnei Ss046] 51 74313729 yrbC hypothetical protein SSON_3340 [Shigella sonnei Ss046] 52 24115498 groEL chaperonin GroEL [Shigella flexneri 2a str. 301] 53 56479605 lpdA dihydrolipoamide dehydrogenase [Shigella flexneri 2a str. 301] 54 24112862 osmE DNA-binding transcriptional activator OsmE [Shigella flexneri 2a str. 301] 55 30065622 deoD purine nucleoside phosphorylase [Shigella flexneri 2a str. 2457T] 56 24111996 sucC succinyl-CoA synthetase subunit beta [Shigella flexneri 2a str. 301] 57 24113762 Crr glucose-specific PTS system component [Shigella flexneri 2a str. 301] 58 24111463 dnaK molecular chaperone DnaK [Shigella flexneri 2a str. 301] 59 74311033 glycoprotein-polysaccharide metabolism 60 30064444 yqjD hypothetical protein S3349 61 82777539 ycbO alkanesulfonate transporter substrate-binding 62 74313684 yraM putative glycosylase 63 24113841 SF2558 OM protein assembly complex subunit YfgL 64 24112186 ybiS hypothetical protein SF0769 65 24111697 tauA taurine transporter substrate binding subunit 66 24115105 yifL putative outer membrane lipoprotein 67 24113718 vacJ lipoprotein precursor 68 1679580 phoN nonspecific phosphatase precursor [Shigella flexneri] 69 13449092 mxiD Type III secretion protein 70 24112703 pspA phage shock protein PspA [Shigella flexneri 2a str. 301] 71 24112822 yeaF hypothetical protein SF1441 [Shigella flexneri 2a str. 301] 72 24113297 SF1963 cystine transporter subunit 73 24113931 SF2652 outer membrane protein assembly complex subunit YfiO [Shigella flexneri 2a str. 301] 74 24114232 sigA serine protease [S. flexneri 2a str. 301] 75 24115037 ATP- F0F1 ATP synthase subunit alpha synt_ab_C 76 24115158 glnA glutamine synthetase [Shigella flexneri 2a str. 301] 77 30061681 aceF dihydrolipoamide acetyltransferase 78 30062108 sucD succinyl-CoA synthetase subunit alpha 79 30062110 sucB dihydrolipoamide succinyltransferase 80 30062117 gltA type II citrate synthase 81 30062179 dacA D-alanyl-D-alanine carboxypeptidase fraction A 82 30062295 glnH glutamine ABC transporter periplasmic protein 83 30062539 agp Glucose-1-phosphate/inositol phosphatase 84 30062760 adhE bifunctional acetaldehyde-CoA/alcohol dehydrogenase 85 30062895 mdoD glucan biosynthesis protein D 86 30062959 gapA glyceraldehyde-3-phosphate dehydrogenase 87 30063091 rspA starvation sensing protein 88 30063194 S1842 bifunctional cysteine desulfurase/selenocysteine lyase 89 30063263 zwf glucose-6-phosphate 1-dehydrogenase 90 30063276 aspS aspartyl-tRNA synthetase 91 30063294 sitA Iron transport protein 92 30063449 yeeX hypothetical protein S2177 93 30063472 hisB imidazole glycerol-phosphate dehydratase/histidinol phosphatase 94 30063593 mglB Galactose-binding transport protein; receptor for galactose taxis 95 30064126 eno phosphopyruvate hydratase 96 30064248 tktA transketolase 97 30064278 ansB L-asparaginase II 98 30064289 S3169 superfamily I DNA helicase 99 30064503 yhbM lipoprotein Nlpl 100 30064729 rpoC DNA-directed RNA polymerase subunit beta′ 101 30064730 rpoB DNA-directed RNA polymerase subunit beta 102 30064872 udp uridine phosphorylase 103 30064882 pldA phospholipase A 104 30064963 atpD F0F1 ATP synthase subunit beta 105 30065048 iutA putative ferric siderophore receptor 106 30065119 lldD L-lactate dehydrogenase 107 30065247 nikA Periplasmic binding proteins for nickel 108 30065291 glpD glycerol-3-phosphate dehydrogenase 109 30065404 rpoA DNA-directed RNA polymerase subunit alpha 110 30065544 hfq RNA-binding protein Hfq 111 56479788 yccZ exopolysaccharide export protein [Shigella flexneri 2a str. 301] 112 56480532 lamB maltoporin [Shigella flexneri 2a str. 301] 113 58045130 sepA SepA [Shigella flexneri] 114 74310732 aceE pyruvate dehydrogenase subunit E1 [Shigella sonnei Ss046] 115 74310771 fhuA ferrichrome outer membrane transporter [Shigella sonnei Ss046] 116 74311118 fepA outer membrane receptor FepA [Shigella sonnei Ss046] 117 74311859 prc Carboxy-terminal protease [Shigella sonni Ss046] 118 74312394 yciD outer membrane protein W [Shigella sonnei Ss046] 119 74312453 prsA ribose-phosphate pyrophosphokinase [Shigella sonnei Ss046] 120 74312677 cirA colicin I receptor [Shigella sonnei Ss046] 121 74312761 glpQ glycerophosphodiester phosphodiesterase [Shigella sonnei Ss046] 122 74312989 talA transaldolase A [Shigella sonnei Ss046] 123 74313764 degQ serine endoprotease [Shigella sonnei Ss046] 124 74314527 malE maltose ABC transporter periplasmic protein [Shigella sonnei Ss046] 125 82543910 SBO_1406 major capsid protein [Shigella boydii Sb227] 126 82544504 ycdO hypothetical protein SBO_2040 [Shigella boydii Sb227] 127 82545484 dsbC thiol:disulfide interchange protein DsbC [Shigella boydii Sb227] 128 82777619 ybjP putative lipoprotein [Shigella dysenteriae Sd197] 129 110807066 yhbN hypothetical protein SFVJ230 [Shigella flexneri 5 str. 8401] 130 161486535 yajG hypothetical protein S0385 131 187427808 tolC outer membrane protein TolC [Shigella boydii CDC 3083-94] 132 187731061 SbBS512_E3369 peptidase, M48B family [Shigella boydii CDC 3083-94] 133 187731375 SbBS512_E3904 outer membrane lipoprotein, Slp family [Shigella boydii CDC 3083-94] 134 187733898 osmY osmotically inducible protein Y [Shigella boydii CDC 3083-94] 135 187734005 bglX beta-glucosidase, periplasmic [Shigella boydii CDC 3083-94] 136 30065453 pepA leucyl aminopeptidase

SEQ ID NOs: 70, 71, 73, 74, 76, 111, 112, 114-129 & 131-135 were identified from S. sonnies ΔtolR blebs. SEQ ID NOs: 8-15 & 17-58 were identified from S. sonnies ΔtolRΔgalU blebs. SEQ ID NOs: 83, 94, 97 & 107 were identified from S. flexneri ΔtolR blebs. SEQ ID NOs: 68, 69, 72, 75, 77-82, 84-93, 95, 96, 98-106, 108-10, 113, 130 & 136 were identified from S. flexneri ΔtolRΔrfbG blebs. SEQ ID NOs: 60-67 were identified from surface digestion of S. sonnies.

Subset 1: SEQ ID NOs: 68, 69, 72, 75, 77-110, 113, 130 & 136.

Subset 2: SEQ ID NOs: 8-15, 17-58, 60-67, 70, 71, 73, 74, 76, 111, 112, 114-129 & 131-135.

Subset 3: SEQ ID NOs: 1-60.

NB: SEQ ID NO: 18 is the same as SEQ ID NO: 5; SEQ ID NO: 33 is the same as SEQ ID NO: 2; SEQ ID NOs: 9 & 16 are related (˜97% identity); SEQ ID NOs: 23 & 59 are related (˜98% identity).

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1. A Shigella bacterium which expresses no more than 4 of TolA, TolB, TolQ, TolR and Pal proteins.
 2. The bacterium of claim 1, wherein the bacterium expresses 4 of the proteins, and not TolR.
 3. A Shigella bacterium which does not express a TolR protein.
 4. A bacterium which is a ΔtolR strain of Shigella.
 5. The bacterium of claim 1, wherein the bacterium does not express a native Shigella lipopolysaccharide.
 6. The bacterium of claim 5, wherein the bacterium does not express a native Shigella O antigen.
 7. The bacterium of claim 4, wherein the strain is a ΔtolRΔgalU strain.
 8. The bacterium of claim 1, wherein a stxA and/or stxB gene is inactivated.
 9. A Shigella bacterium which expresses TolA, TolB, TolQ, TolR and Pal proteins, wherein the TolA, TolQ, TolR and/or Pal protein (a) is located in the bacterium's inner or outer membrane, and (b) includes one or more amino acid sequence mutation(s) such that, compared to the same bacterium without said mutation(s), the bacterium releases greater quantities of outer membrane blebs when growing in culture medium.
 10. A Shigella bacterium in which one or more components of its Tol-Pal system has a modification such that, during growth in culture medium, the bacterium releases greater quantities of outer membrane blebs into the medium than the same bacterium lacking the modification, and which does not express: (i) a native Shigella lipopolysaccharide and/or (ii) a Shigella enteric toxin.
 11. A process for preparing Shigella blebs, comprising a step of separating the blebs from a culture medium comprising a bacterium of claim 1, which have been grown under conditions which permit the release of blebs into the medium by the bacteria.
 12. The process of claim 11, wherein the bacteria have been grown under iron-limiting conditions.
 13. A method of preparing a hyperblebbing Shigella bacterium, comprising a step of modifying gene(s) encoding one or more components of a starting bacterium's Tol-Pal system such that the modification causes the bacterium, when grown in culture medium, to release greater quantities of outer membrane blebs into the medium than the starting bacterium, and wherein the modification involves mutating one or more of the starting bacterium's TolA, TolB, TolQ, TolR and/or Pal genes.
 14. A bleb isolated or obtainable from a bacterium of claim
 1. 15. A composition comprising blebs that, during culture of a bacterium of claim 1 are released into the culture medium.
 16. The composition of claim 15, which does not comprise any living and/or whole bacteria.
 17. A composition comprising blebs, wherein the blebs are present in the filtrate obtainable after filtration through a 0.22 μm filter of a culture medium in which a bacterium of claim 1 has been grown.
 18. Culture media comprising bacteria of claim 1, which have been grown under conditions which permit the release of blebs into the medium by the bacteria.
 19. A Shigella strain which does not express a functional HtrB enzyme.
 20. The Shigella strain of claim 19, which is a htrB knockout.
 21. A Shigella bleb which includes one or more of: (a) a protein consisting of an amino acid sequence selected from SEQ ID NOs: 8 to 67; (b) a protein comprising an amino acid sequence having at least 85% identity to one of SEQ ID NOs: 8 to 67 and/or comprising a fragment of at least 7 consecutive amino acids of any one of SEQ ID NOs: 8 to 67, wherein the fragment comprises an epitope.
 22. A bleb-free immunogenic composition comprising a protein comprising: (a) amino acid sequences SEQ ID NOs 8 to 136; (b) an amino acid sequence having at least 85% identity to one of SEQ ID NOs: 8 to 136 and/or comprising a fragment of at least 7 consecutive amino acids of any one of SEQ ID NOs: 8 to 136 and comprising an epitope from one of SEQ ID NOs: 8 to
 136. 23. A process for culturing Shigella, wherein iron-availability is limiting.
 24. A bleb isolated or obtainable from a bacterium obtainable by the method of claim
 13. 25. A bleb isolated or obtainable by the process of claim
 11. 26. A composition comprising blebs obtainable by the method of claim 13 that are released into the culture medium.
 27. A composition comprising blebs, wherein the blebs are present in the filtrate obtainable after filtration through a 0.22 μm filter of a culture medium obtainable by the method of claim 13 has been grown.
 28. Culture media comprising bacteria obtainable by the method of claim 13, which have been grown under conditions which permit the release of blebs into the medium by the bacteria. 